On-Demand Release Of Persulfides As Antioxidants

Cellular redox balance is critical for maintaining normal function and preventing damage to DNA and proteins, which may lead to accelerated aging, and, more nefariously, the development of Alzheimer’s disease, heart failure, depression, and others. Generation and sale of antioxidant supplements is a multi-billion dollar industry, despite apparent discrepancies in efficacy, mode of action, and effects on long-term health of many of these supplements.

While it is generally recognized that preserving cellular redox balance and preventing a high concentration of reactive oxygen species (ROS, i.e., free radicals) is crucial to maintaining healthy organ and systems, scientists are still debating the specific species responsible for maintaining this balance. In this context, the importance of molecules containing sulfur as essential cellular signaling molecules has emerged within the last decade. Research into sulfur compounds as necessary components for normal cellular function in mammals began with hydrogen sulfide (H2S), which had long been known only as a smelly, toxic gas produced naturally from deep inside the earth or as an industrial byproduct. As it turns out, H2S is produced throughout the body and is responsible for mediating a wide variety of cellular processes. As our understanding of the various roles of H2S in the body has expanded, we have begun discovering other sulfur-containing molecules which likely have vital cellular functions.

Among these biologically-active sulfur species are persulfides (R–SSH). Persulfides were initially believed to only form on proteins as a means of easily modulating the protein’s activity. However, recent research has demonstrated that the same enzymes responsible for producing H2S produce free persulfides (persulfides not appended to proteins) enzymatically in our cells. What makes persulfides particularly interesting is the wide variety of chemical reactions they can partake in, owing to the ability of sulfur to occupy eight oxidation states. Previous studies on persulfide chemistry, while limited, have demonstrated that persulfides have a greater reducing potential (greater antioxidant behavior) than most other sulfur species identified in cells. Taken altogether, our knowledge that persulfides are generated enzymatically in cells, combined with their enhanced reducing potential, indicates that persulfides may be key players in maintaining cellular redox balance.

Unfortunately, the same chemical properties that enable persulfide signaling in cells also hinder our ability to study them. Previous attempts to synthesize persulfides in the lab have led to unstable compounds that decompose rapidly when isolated.

This paper describes a class of compounds conceptualized as “protected persulfides,” which can be readily isolated in the lab. These protected persulfides are stable at room temperature under normal storage conditions, which alleviates the challenges with isolation and handling previously encountered when studying persulfides. These protected persulfides release the desired free persulfide species only in response to a specific, biologically relevant stimulus. The first iteration of these compounds, termed BDP-NAC, is selectively responsive to hydrogen peroxide (H2O2), a common ROS found in the body under hyperoxidative conditions, similar to those after a heart attack.

Once the protected persulfide, BDP-NAC, was successfully synthesized, a number of experiments were conducted to measure the timescale of release and selectivity of BDP-NAC towards other compounds that may potentially trigger persulfide release. Analysis showed that the expected persulfide was released over the course 2 hours — a biologically relevant timescale. Additional experiments showed that persulfide release occurred selectively in the presence of H2O2 over other common biological oxidants.

Finally, cell studies were conducted using mice cardiac muscle cells. As a negative control, cells treated with H2O2 showed a 70% reduction in viability, mimicking the cell death that occurs after cardiovascular trauma. Excitingly, BDP-NAC delivered to the cells concurrently with H2O2 eliminated the deleterious effects of H2O2. Additionally, BDP-NAC showed greater efficacy in protecting cells against H2O2 than common H2S-releasing compounds and other relevant controls. This is an exciting demonstration of the capabilities of persulfides to act as antioxidants, lending further evidence in support of their importance in maintaining cellular redox balance.